Optimization Of Transient Protein Expression In CHO Cells

By Peter O’Callaghan, Head of Expression System Sciences, Biologics and Licensing, Lonza

Transient transfection is a relatively low-cost, rapid method for producing recombinant proteins, but generally provides fairly low titers as expression is predominately episomal and no selection for plasmid transfection is applied. By contrast, stable pools are generated following transfection by applying selection pressure so that only cells with integrated DNA can grow. The selection process adds significant time to the development timeline, but higher titers can be achieved.

Regardless of the method, to triage therapeutic candidates early in development, recombinant proteins produced during discovery and early-phase development must have product quality attributes (PQAs), such as isoform composition, percent aggregates and post-translational modifications, broadly comparable to material produced using a stable cell line. This is a particular concern when using alternative cell lines such as HEK for transient expression before moving to a Chinese hamster ovary (CHO) host for stable expression.

Typically, stable CHO cell pools created using transposase systems such as GS piggyBac^® support significantly higher titers than those obtained from transient transfections. They also have the additional advantage of supporting cryopreservation, thereby enabling future rounds of product generation upon revival. However, the longer timeline associated with generating stable pools means that transient expression will usually be selected when speed is of the essence. To close the titer gap with stable pools and more fully realize the speed advantage of transient expression, optimization of the transient expression workflow is required.

An ideal solution would offer speed and also afford high titers of recombinant proteins with PQAs comparable to those generated using stable processes (we deem these PQAs ‘CHO-like’). At Lonza we have been optimizing the stable pool expression format in recent years, particularly since the introduction of the GS piggyBac^® hyperactive transposase technology. We therefore sought to apply the learnings from that work to create an optimized transient expression process that merges the best attributes of both traditional transient and stable pool expression.

The new GS Discovery^® solution from Lonza, part of the GS Gene Expression System^®, meets this need by offering such a hybrid process. It is to our knowledge the first example wherein a transposase enzyme has been used to boost titers from a transient transfection process over such a short timeframe. The GS Discovery^® solution also includes a standard transient process for applications where speed and low cost are most important. Both leverage the high-performing GS Xceed^® CHOK1SV GS-KO^® cells.